Vrije Universiteit Brussel

Structural insights into binding specificity, efficacy and bias of a 2AR partialagonistMasureel, Matthieu; Zou, Yaozhong; Picard, Louis-Philippe; van der Westhuizen, Emma;Mahoney, Jacob P; Rodrigues, João P G L M; Mildorf, Thomas J; Dror, Ron O; Shaw, DavidE; Bouvier, Michel; Pardon, Els; Steyaert, Jan; Sunahara, Roger K; Weis, William I; Zhang,Cheng; Kobilka, Brian KPublished in:Nature Chemical Biology

DOI:10.1038/s41589-018-0145-x

Publication date:2018

License:Unspecified

Document Version:Final published version

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Citation for published version (APA):Masureel, M., Zou, Y., Picard, L-P., van der Westhuizen, E., Mahoney, J. P., Rodrigues, J. P. G. L. M., ...Kobilka, B. K. (2018). Structural insights into binding specificity, efficacy and bias of a

2AR partial agonist.

Nature Chemical Biology, 14(11), 1059-1066. https://doi.org/10.1038/s41589-018-0145-x

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Articleshttps://doi.org/10.1038/s41589-018-0145-x

1Department of Molecular and Cellular Physiology, Stanford University School of Medicine, Stanford, CA, USA. 2Geneus Technologies, Ltd, Chengdu, Sichuan, People’s Republic of China. 3Department of Biochemistry, Institute for Research in Immunology and Cancer, Université de Montreal, Montreal, Québec, Canada. 4Department of Pharmacology, University of Michigan, Ann Arbor, MI, USA. 5Department of Computer Science, Stanford University, Stanford, CA, USA. 6Department of Structural Biology, Stanford University, Stanford, CA, USA. 7D. E. Shaw Research, New York, NY, USA. 8Department of Biochemistry and Molecular Biophysics, Columbia University, New York, NY, USA. 9Structural Biology Brussels, Vrije Universiteit Brussel, Brussels, Belgium. 10Structural Biology Research Center, VIB, Brussels, Belgium. 11Department of Pharmacology, University of California San Diego School of Medicine, La Jolla, CA, USA. 12Department of Pharmacology and Chemical Biology, University of Pittsburgh School of Medicine, Pittsburgh, PA, USA. 13Present address: Monash Institute for Pharmaceutical Sciences, Monash University, Victoria, Australia. 14Present address: Department of Molecular and Cellular Physiology, Stanford University School of Medicine, Stanford, CA, USA. 15Present address: Dropbox, New York, NY, USA. 16Present address: Department of Computer Science and Institute for Computational and Mathematical Engineering, Stanford University, Stanford, CA, USA. 17These authors contributed equally: Matthieu Masureel, Yaozhong Zou. *e-mail: chengzh@pitt.edu; kobilka@stanford.edu

G-protein-coupled receptors (GPCRs) are able to respond to a large variety of ligands with different efficacy profiles for specific signaling pathways1: full agonists that maximally

stimulate; partial agonists that produce submaximal stimulation even at fully saturating concentrations; inverse agonists that sup-press basal signaling; and neutral antagonists that bind the receptor without stimulating or inhibiting basal signaling. Among these cat-egories, partial agonists are often better tolerated than full agonists as therapeutics2.

Salmeterol is a partial agonist for the human β 2AR. As a potent bronchodilator, it is among the most prescribed drugs for the treatment of asthma and chronic obstructive pulmonary disease (COPD)3. Compared with some other β 2AR agonists such as iso-proterenol and salbutamol, salmeterol has two desirable pharmaco-logical properties. First, salmeterol is able to distinguish β 2AR from β 1AR for selective stimulation (1,400- to 3,000-fold selectivity)4, thereby minimizing cardiac toxicity5. Second, salmeterol belongs to the class of long-acting β 2AR agonists (LABAs), which have a dura-tion of action up to 12 h, in contrast to the short-acting β 2AR ago-nists, such as salbutamol, which have only a 4- to 6-h duration of action6–8. Those pharmacological properties of salmeterol have con-tributed to its successful use in treating asthma and COPD for more than two decades. However, LABAs, especially salmeterol, when used alone, have been suggested to be associated with increased

mortality in asthmatics in several clinical trials. This liability is miti-gated when LABAs are combined with an inhaled corticosteroid9–11.

The high selectivity and long-acting properties of salmeterol have been attributed to its unusual bitopic structure. In addition to the saligenin ethanolamine pharmacophore that replaces the catechol-amine structure of the endogenous β 2AR ligand epinephrine (also known as adrenaline), salmeterol contains an additional moiety of an aryloxyalkyl tail consisting of a phenol ring with an 11-atom ether chain (Fig. 1a). While the pharmacophore binds the orthosteric site, which is responsible for receptor activation, the aryloxyalkyl tail has been proposed to bind to an additional site (exosite), thus allowing for additional interactions responsible for the high receptor-subtype selectivity and the slow dissociation rate contributing to its long duration of action12. Previous mutagenesis and biochemical stud-ies seeking to locate the exosite have yielded conflicting results4,12–16. Salmeterol is also of interest because it is a functionally selective β 2AR partial agonist with a 5- to 20-fold bias toward Gs over arres-tin17,18. Previous studies have revealed that activation of the β 2AR by salmeterol, compared with isoproterenol, leads to slower initial rates of G-protein-coupled receptor kinase (GRK) phosphorylation and similar maximal degrees of phosphorylation19,20, but strongly decreased arrestin-mediated receptor internalization and desensiti-zation17,21,22, thus contributing to the prolonged therapeutic effect of salmeterol in bronchial dilation as a result of β 2AR stimulation. This

Structural insights into binding specificity, efficacy and bias of a β2AR partial agonistMatthieu Masureel1,17, Yaozhong Zou1,2,17, Louis-Philippe Picard3, Emma van der Westhuizen   3,13, Jacob P. Mahoney4,14, João P. G. L. M. Rodrigues1,5,6, Thomas J. Mildorf7,15, Ron O. Dror7,16, David E. Shaw7,8, Michel Bouvier   3, Els Pardon9,10, Jan Steyaert9,10, Roger K. Sunahara   11, William I. Weis1,6, Cheng Zhang   12* and Brian K. Kobilka1*

Salmeterol is a partial agonist for the β 2 adrenergic receptor (β 2AR) and the first long-acting β 2AR agonist to be widely used clinically for the treatment of asthma and chronic obstructive pulmonary disease. Salmeterol’s safety and mechanism of action have both been controversial. To understand its unusual pharmacological action and partial agonism, we obtained the crystal structure of salmeterol-bound β 2AR in complex with an active-state-stabilizing nanobody. The structure reveals the location of the salmeterol exosite, where sequence differences between β 1AR and β 2AR explain the high receptor-subtype selectivity. A structural comparison with the β 2AR bound to the full agonist epinephrine reveals differences in the hydrogen-bond network involving residues Ser2045.43 and Asn2936.55. Mutagenesis and biophysical studies suggested that these interactions lead to a distinct active-state conformation that is responsible for the partial efficacy of G-protein activation and the limited β -arrestin recruitment for salmeterol.

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signaling bias may contribute to the advantageous therapeutic profile of salmeterol by maintaining bronchodilation through Gs-mediated signaling while minimizing arrestin-mediated β 2AR desensitization and avoiding arrestin-dependent proinflammatory effects23,24.

Recent progress in the structural characterization of β adrenergic receptors, especially the crystal structures of active β 2AR bound to the full agonists BI-167107 or epinephrine, with either a Gs protein or active-state-stabilizing nanobody, as well as the crystal structures of inactive avian β 1AR bound to a variety of full and partial agonists, have greatly advanced understanding of the pharmacology and activation of β adrenergic receptors25–29. To further understand the molecular basis for the unusual pharmacological properties of salmeterol and its partial agonism, we obtained the crystal structure of salmeterol-bound human β 2AR in an active state stabilized by a conformation-specific nanobody, Nb71. The structure reveals the location of the exosite and provides a structural explanation for the high receptor-subtype selectivity and distinct signaling behavior of salmeterol.

ResultsNanobody Nb71 stabilizes salmeterol-bound β2AR. For the β 2AR and the µ -opioid receptor, agonists alone are insufficient to stabilize the receptors in active conformational states27,30–32. As a result,

the active-state structures of the β 2AR and µ -opioid receptor have required either a G protein or conformation-specific camelid anti-body fragments (nanobodies) to stabilize the active states of the receptors25,26,29. The first active-state structure of a hormone-acti-vated GPCR was obtained with a nanobody named Nb80, which was obtained from a llama immunized with β 2AR bound to the ultra-high-affinity full agonist BI-167107 reconstituted into phospholipid vesicles25. The structures of β 2AR in the β 2AR–Nb80 complex and the β 2AR–Gs complex are very similar25,26. However, evidence from biophysical studies suggests that partial agonists may stabilize dis-tinct states33; consequently, Nb80 may not be the best candidate to stabilize salmeterol-bound β 2AR.

Whereas Nb80 preferentially binds agonist-occupied β 2AR rather than antagonist-occupied β 2AR25, Octet RED studies indicated that Nb80 also preferentially binds β 2AR bound to BI-167107 and epi-nephrine rather than β 2AR bound to salmeterol (Supplementary Table 1). Moreover, Nb80 has a greater effect in enhancing the binding affinity of the agonist isoproterenol than the partial agonist salmeterol (Supplementary Fig. 1). We therefore selected another nanobody, Nb71, generated from the same immunization that pro-duced Nb80. Nb71 preferentially binds agonist-occupied β 2AR34, but, in contrast to Nb80, it has no preference for these catecholamines

c

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Fig. 1 | Crystal structure of salmeterol-bound β2AR. a, Chemical structures of the following β 2AR ligands: partial agonists salmeterol and salbutamol; full agonists epinephrine and BI-16707. The respective pharmacophores that bind the orthosteric ligand-binding pocket are highlighted in purple, and the aryloxyalkyl tail of salmeterol, which binds the exosite, is highlighted in blue. b, Overall ribbon representation of the salmeterol-bound β 2AR–Nb71 complex. The T4L lysozyme fusion facilitates crystallization, and Nb71 stabilizes the active, salmeterol-bound (orange spheres) β 2AR. c, Cross-section through the receptor, with the interior in black, highlighting salmeterol (orange spheres) occupying the orthosteric site and the exosite.

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over salmeterol (Supplementary Table 1 and Supplementary Fig. 1). Therefore, we chose Nb71 to stabilize the salmeterol-bound β 2AR for structural characterization.

Structural features of Nb71-stabilized β2AR. We used an engi-neered β 2AR with T4 lysozyme (T4L) fused to its N-terminal region and a truncated intracellular loop3 (referred to as T4L-β 2AR-Δ ICL3 hereafter) to crystallize β 2AR bound to salmeterol. Previous studies have demonstrated that T4L-β 2AR-Δ ICL3 exhibits similar ligand binding and G-protein-activating properties to those of the wild-type β 2AR35. Using this construct, we obtained crystals of the β 2AR–salmeterol–Nb71 complex through the lipidic cubic phase method36. A complete dataset was obtained by merging data from 23 crystals, and the structure was determined to a resolution of 3.0 Å (Fig. 1b,c and Supplementary Table 2). Like Nb80, Nb71 binds the cytoplasmic surface of the receptor with its third complemen-tarity-determining-region loop inserted into a hydrophobic cavity formed by residues from transmembrane helices (TMs) 3, 5, 6 and 7 (Fig. 1b and Supplementary Fig. 2), yet Nb71 stabilizes a conforma-tion distinct from that stabilized by Nb80 (Supplementary Fig. 2).

The active-state conformation of the receptor in the β 2AR–sal-meterol–Nb71 complex resembles that of the BI-167107-bound β 2AR in complex with Nb80 (PDB 3P0G, r.m.s. deviation 1.0 Å) or with Gs (PDB 3SN6, r.m.s. deviation 1.3 Å) more than the inactive-state conformation of the receptor in the structure of β 2AR bound to the inverse agonist carazolol (PDB 2RH1, r.m.s. deviation 1.9 Å)25,26. Indeed, the structural features associated with receptor activation25, including the outward movement of TM6 at the cytoplasmic side associated with the conformational changes of the core triad resi-dues Pro2115.50, Ile1213.40 and Phe2826.44 (Ballesteros–Weinstein numbering), as well as the slightly contracted ligand-binding pocket at the extracellular side, are all present in the structure of the β 2AR–salmeterol–Nb71 complex, as compared with the inactive β 2AR (Fig. 2a–c). However, the Nb71-stabilized β 2AR shows some struc-tural features distinct from those of the Nb80- and Gs-stabilized β 2AR. Relative to the inactive β 2AR, the Nb71-stabilized β 2AR dis-plays a smaller outward movement of the cytoplasmic end of TM6 (8 Å) than the Nb80-stabilized β 2AR (11 Å) or Gs-stabilized β 2AR

(13 Å) (Fig. 2a). There is also a slightly smaller counterclockwise rotation of TM6 in the Nb71-stabilized β 2AR when viewed from the cytoplasmic surface, as shown by the position of Glu268 (Fig. 2a). Although this current structure clearly shows an active-state con-formation of β 2AR, the smaller conformational rearrangements observed for TM6 after activation, as compared with the changes seen for the active-state-stabilized β 2AR bound to Nb80 or Gs, sug-gest a less active or a partially active conformational state of β 2AR stabilized by Nb71. This partially active conformational state might potentially closely resemble a salmeterol-stabilized conformation of β 2AR that is less efficient at coupling to Gs. However, the conforma-tion of TM6 is primarily stabilized by interactions with Nb71 and may not reflect the conformation stabilized by salmeterol alone.

Exosite binding of salmeterol. The location and the molecular details of the exosite for the aryloxyalkyl tail of salmeterol are of great interest because of its association with the high receptor selec-tivity, high affinity and long-lasting action of salmeterol. The clear electron density map of salmeterol based on our structure allowed us to unambiguously define the structural basis of the exosite (Fig. 3a,b and Supplementary Figs. 3 and 4). The crystal structure shows that the aryloxyalkyl tail of salmeterol extends toward the extracel-lular surface of the receptor, occupying a cleft formed by residues from extracellular loop (ECL) 2, ECL3 and the extracellular ends of TM6 and TM7. Interactions between salmeterol and the exosite are mediated primarily through extensive van der Waals and hydro-phobic interactions (Fig. 3c). The phenol ring of the tail also forms π –π interactions with the surrounding aromatic residues Phe194ECL2, Tyr3087.35 and His2966.58, in agreement with previous studies report-ing a 5- to 18-fold decrease in salmeterol affinity caused by muta-tion of those residues4. In addition, the ether oxygen atom of the tail forms a hydrogen bond with the main chain amide group of residue Phe193ECL2 (Fig. 3a). This ether oxygen atom acts as a ‘hinge’ point where the tail of salmeterol bends almost 90° to fit the exosite. Previous studies have indicated that shifting or removing the ether oxygen in the aryloxyalkyl tail substantially decreases the affinity of salmeterol for β 2AR21, thus suggesting an important role of this hydrogen bond in exosite binding (Supplementary Fig. 5). All those

TM5

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a b c

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BI-167107–β2AR Salmeterol–β2AR

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Ser207

Phe282

Ile121

Pro211

Fig. 2 | Structural features of the β2AR–salmeterol–Nb71 complex. a–c, Overlay of inactive carazolol-bound receptor (gray), Nb71- and salmeterol-bound β 2AR (blue), and BI-167107-bound β 2AR–Gs complex (green and pink, respectively). a, Side view (inset) and cytoplasmic view of TM6, showing the position of TM6 relative to the helical bundle. The extent of TM6 movement, indicated by black arrows, was measured with the Cα of Glu268 as a reference point. b, Rearrangement of Pro2115.50, Ile1213.40 and Phe2826.44 and the associated outward movement of the cytoplasmic end of TM6. The inactive structure of β 2AR is gray. The active structures of β 2AR are blue (coupled with salmeterol and Nb71), cyan (coupled with epinephrine and 6B9) and green (coupled with BI-167107 and Gs). c, Extracellular (top) view of β 2AR, with major conformational changes among structures indicated by red arrows and ligands shown in stick representation.

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additional interactions in the exosite contribute to the 1,000-fold-higher affinity of salmeterol over salbutamol, a short-acting β 2AR agonist that shares the same orthosteric pharmacophore as salme-terol but lacks the long aryloxyalkyl tail37 (Fig. 1a).

The exosite binding also explains the very high selectivity of sal-meterol for β 2AR over β 1AR (> 3,000 fold)37. β 1AR and β 2AR share a very high overall structural similarity (r.m.s. deviation of carazolol-bound avian β 1AR and human β 2AR = 0.58 Å; PDB 2YCW and PDB 2RH1, respectively) and sequence similarity (92% in TM segments for human β 1AR and β 2AR; Supplementary Fig. 6). In particular, all residues that form the orthosteric binding pocket, with the exception of Tyr308, are identical in β 1AR and β 2AR. In contrast, the exosite is relatively divergent in those two receptors. Salmeterol achieves high receptor selectivity by sampling this divergent region with its long aryloxyalkyl tail (Fig. 3d and Supplementary Fig. 6). Such selectivity determinants do not apply to salbutamol, which has only approxi-mately 20-fold selectivity for β 2AR over β 1AR37. Our results are in agreement with extensive chimeric-receptor and site-directed-muta-genesis studies recently reported by Baker et al.4, who have observed that the K305D and H296K mutations in the β 2AR substantially affect salmeterol affinity but have little effect on salbutamol affinity.

The salmeterol exosite in β 2AR is reminiscent of the well-defined allosteric site in the M2 muscarinic receptor (M2R)38. The M2R is one of the most extensively characterized model systems for alloste-ric regulation. We compared the exosite in the β 2AR and the bind-ing site for the positive allosteric modulator LY2119620 (Fig. 3e), as revealed by the active-state crystal structure of M2R bound to the orthosteric agonist iperoxo and LY2119620 (ref. 38). The similarity between those two sites suggests the potential for allosteric regu-lation of β 2AR activity by small molecules that target the exosite. However, the bitopic nature of salmeterol with two-site binding on β 2AR supports the feasibility of developing highly selective bitopic compounds for other GPCRs including muscarinic receptors39.

Polar interactions within the orthosteric binding site. Although the interactions between the aryloxyalkyl tail of salmeterol and

the exosite in the extracellular vestibule of β 2AR confer the high affinity and selectivity of salmeterol, they are not responsible for its efficacy and signaling bias. Salmeterol and salbutamol exhibit similar signaling properties including partial agonism and the selective activation of Gs over arrestin and Gi17 (Supplementary Fig. 7), despite their differences in affinity and receptor selectiv-ity. Given that salmeterol and salbutamol share only the saligenin ethanolamine pharmacophore (Fig. 1a), the interactions between this shared pharmacophore and the receptor within the orthosteric binding pocket are likely to be responsible for their shared signal-ing properties. In the orthosteric binding site, the alkylamine and β -hydroxyl groups of salmeterol form hydrogen-bonding interac-tions with Asp1133.32 and Asn3127.39, and the saligenin group forms hydrogen-bonding interactions with Ser2035.42 and Ser2075.46, which are similar to the interactions observed for β 2AR bound to the full agonist epinephrine (Fig. 4a,b). In the structure of β 2AR bound to epinephrine, the hydrogen-bonding interactions with Ser2035.42 and Ser2075.46 are associated with the inward movement of TM5 around those two serine residues, which is further linked to the rearrange-ment of the core triad residues and the outward movement of the cytoplasmic end of TM6 (ref. 29). For salmeterol, the two saligenin hydroxyl groups also form direct hydrogen-bonding interactions with Ser2035.42 and Ser2075.46. However, these interactions with sal-meterol, compared with those with epinephrine, may have a weaker effect on stabilizing the inward movement of TM5 because of the additional methylene between the meta-position hydroxyl group and the phenyl ring.

In epinephrine-bound β 2AR, Asn2936.55 forms a hydrogen bond with Ser2045.43 and the meta-hydroxyl of epinephrine. This polar network is not observed in the salmeterol-bound β 2AR. Asn2936.55 and Ser2045.43 have previously been shown to be important for the binding of epinephrine but not for the binding of antagonists40,41. To further investigate the possible role of these polar networks in ligand efficacy, we used molecular dynamics (MD) simulations to charac-terize the stability of specific ligand–receptor hydrogen bonds that would be expected to stabilize the inward movement of TM5 and

F194

K305

H296

Y308

I309

d

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H296

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S207

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LY2119620

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β2AR M2 muscarinic

TM7

TM7

Fig. 3 | Salmeterol exosite and receptor-subtype-selectivity determinants. a,b, Side (a) and top (b) views of β 2AR, with stick representation of salmeterol (orange). c, Spherical representation of the salmeterol aryloxyalkyl moiety (orange) and the β 2AR (blue). d, Residues important for interaction and specificity are highlighted in yellow and labeled. With the exception of Y308 (indicated by a red arrow), the amino acids that form the orthosteric binding pocket for epinephrine are identical between β 1AR and β 2AR. e, Comparison of the ligand-binding sites between β 2AR (left) bound to salmeterol (orange sticks) and M2R (right) bound to the allosteric modulator LY2119620 (green sticks).

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the rotation of TM6 observed in active-state structures of the β 2AR (Fig. 5a). These simulations show that salmeterol forms less stable hydrogen bonds with Asn2936.55 and Asn3127.39 than does epineph-rine. Salmeterol forms a more stable hydrogen bond with Ser2035.42, apparently only because its longer hydroxymethyl group can main-tain this hydrogen bond better than epinephrine’s hydroxyl group, even when TM5 moves away from its crystallographic position. Thus, although the salmeterol-bound crystal structure captured a binding-pocket conformation similar to that observed for the full agonist, MD simulations suggest that this active conformation is stabilized less by salmeterol than by epinephrine, and the most dramatic dif-ference was observed for Asn2936.55. To further characterize the role

of Asn2936.55 and Ser2045.43 interactions, we examined the effects of mutating these residues on β -arrestin2 recruitment and Gs activa-tion by using bioluminescence resonance energy transfer (BRET) between membrane-anchored GFP and luciferase-tagged arrestin or luciferase-tagged Gs and GFP-tagged Gβ1/Gγ1, respectively42,43 (Fig. 5b,c and Supplementary Table 3). For all the mutations tested in the presence of isoproterenol, a catecholamine similar to epineph-rine, we observed a dramatic decrease in β -arrestin2 recruitment (Fig. 5b) and a more moderate decrease in Gs activation (Fig. 5c). This result suggests an important role of these two residues in regulating bias, either by directly modulating arrestin coupling or by altering GRK phosphorylation and hence arrestin binding.

Asn312

Asp113Asp113

TM5

TM7

TM6

TM3

Asn293

Ser203Ser204

Ser207

β2AR with salmeterolβ2AR with epinephrine

Asn312

TM5

TM7

TM6

TM3

Asn293

Ser203

Ser204

Ser207

a b

Fig. 4 | hydrogen-bonding interactions in the orthosteric site and rearrangement of the ligand-binding pocket. a,b, Comparison of ligand-mediated hydrogen bonds (black dashed lines) in epinephrine-bound (a) and salmeterol-bound (b) β 2AR.

β-arrestin2 recruitment β-arrestin2 recruitment

–10 –9 –8 –7 –6 –5 –4

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Fig. 5 | Ligands and specific residues in the orthosteric site modulate hydrogen-bonding and signaling outcome. a, Frequency of hydrogen-bonding between Ser2035.42 and Asn2936.55 and the ligand m-OH group; Ser2075.46 and the ligand p-OH group; and Asn3127.39 and the ligand β -OH group in MD simulations of receptor bound to epinephrine or salmeterol. Individual data points (black dots) and s.e.m. (error bars) of 3 independent experiments are shown. Statistical significance was assessed with one-sided Welch’s unequal variance t test (P values: ****Ser2035.42 =  0.00003; Ser2075.46 =  0.08 (nonsignificant, NS); **Asn2936.55 =  0.004; **Asn3127.39 =  0.01). The actual statistical significance of the differences (for example, for Ser207) may be higher than computed, because each of the data points is based on a trajectory with thousands of samples. b,c, BRET-based assays monitoring β -arrestin2 recruitment (b) and G-protein activation (c) on wild-type (WT) and the indicated mutant β 2AR constructs. Data represent the mean (symbols) ±  s.e.m. (error bars) of 3 independent experiments. Error bars shorter than the height of the symbol are not shown.

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Interestingly, the same mutations, with the exception of S204T, had a less pronounced effect on the ability of salmeterol to activate Gs (Fig. 5c), and all mutants showed very little or no salmeterol-induced arrestin recruitment, similarly to wild-type β 2AR (Fig. 5b). Thus, the absence of the hydrogen bonds between Asn2936.55 and salmeterol may account for the weak β -arrestin recruitment as well as the lower efficacy in Gs activation of salmeterol-bound β 2AR compared with β 2AR bound to full agonist.

Spectroscopic insights into salmeterol-bound β2AR. The dif-ferences in efficacy and signaling bias between salmeterol and the full agonist epinephrine suggest that salmeterol may stabilize a dis-tinct conformation. Previous single-molecule Förster resonance energy transfer (FRET) studies have suggested a smaller outward displacement of TM6 in β 2AR bound to salmeterol compared with β 2AR bound to epinephrine or BI167107 (ref. 33). In addition, cellular assays using a BRET-based β 2AR conformational sensor44 confirmed salmeterol’s lower propensity to promote the outward movement of TM6 (Supplementary Fig. 8). Nevertheless, these FRET studies were unable to distinguish the mechanistic possibilities of whether salme-terol either stabilizes the same conformation of TM6 as epinephrine but does so for a smaller receptor population, or stabilizes a distinct TM6 conformation. To further investigate the differences at the cyto-plasmic end of TM6 after activation between salmeterol-bound and epinephrine-bound β 2AR in the absence of conformation-stabilizing nanobodies, we performed steady-state spectroscopic studies on purified, labeled receptor in detergent with two different reporter systems (Supplementary Fig. 9a). We used β 2AR labeled at Cys265 with the fluorophore monobromobimane (bimane or mBBr), which we previously used as a conformational reporter of β 2AR activa-tion31,45, and developed a new fluorescent reporter system using an engineered β 2AR labeled at residue 266 with the fluorophore Atto655.

The bimane fluorophore has been extensively used to report on the activation of β 2AR25,45,46 as well as other GPCRs47,48, because it

is very sensitive to its chemical environment. In the inactive state, bimane attached at the cytoplasmic end of TM6 is in a hydropho-bic environment. After receptor activation, the intracellular end of TM6 undergoes an outward and ‘unwinding’ motion (Fig. 6a) that shifts bimane to a more polar and solvent-exposed environment, thus resulting in a decrease in fluorescence intensity and a redshift in wavelength of maximum emission intensity (λmax). Agonists alone produced a 10–20% decrease in intensity and an ∼ 10-nm redshift in λmax. Further changes were observed after G-protein coupling. Using this reporter system, we found that even though salmeterol has a higher binding affinity than epinephrine to β 2AR6, epinephrine causes a larger decrease than salmeterol in the intensity and redshift in λmax of bimane when both ligands are used at saturating concen-trations (Fig. 6b). This result suggests that salmeterol stabilizes β 2AR in conformations with on average a smaller outward movement or rotation of TM6. In addition, we observed that Gs induced similar bimane responses with salmeterol and epinephrine (Fig. 6b; ‘+ Gs’ curves), thus indicating that, once coupled to Gs, the β 2AR adopts a similar TM6 conformation regardless of which agonist is bound at the extracellular region, in agreement with previous single-molecule FRET studies33. Nevertheless, the extent to which the salmeterol-bound receptor couples to Gs appears to be slightly lower, because the change in λmax and fluorescence intensity is slightly less pro-nounced than that for the epinephrine-bound receptor.

To complement the bimane-based measurements, we developed a new distance-sensitive fluorescent reporter. Rather than relying on FRET, in which stoichiometric and specific labeling of donor and accep-tor is required to faithfully report on distance changes in bulk measure-ments, we used a single-dye reporter system. In photoinduced electron transfer (PET), fluorophores such as Atto655 can be quenched by a tryptophan in proximity, through the formation of weakly fluorescent or nonfluorescent dye–tryptophan ground-state complexes49 (Fig. 6c). Owing to the strong distance dependence of PET, small-scale con-formational changes result in ‘on/off ’ switching of fluorescence, thus

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Fig. 6 | Spectroscopic interrogation of ligand-induced changes in TM6 conformation on detergent-solubilized, purified labeled receptor. a,c. Schematic representation of labeled receptor constructs used to probe the outward motion of TM6. The β 2AR transmembrane helices 1–5 and 7 are shown as gray cylinders. The blue cylinders represent TM6 in the inactive (light blue) and active (dark blue) conformations, with the labels attached at the intracellular end of TM6 depicted according to their overall structure. The bimane fluorophore (teal) is an environment-sensitive reporter, whereas the Atto655 dye (pink) is quenched in a distance-dependent manner by an engineered tryptophan (L230W, yellow). b,d, Steady-state fluorescence emission spectra of bimane-labeled (b) and Atto655-labeled (d) receptor in the presence of epinephrine (epi) or salmeterol (salm). The spectra are normalized relative to ligand-free receptor (apo; light-blue curve) and show the fluorescence dose–response curves of receptors up to saturating ligand concentrations, in the absence or presence of G protein. Data are plotted as mean (curves) ±  s.d. (error bars) of triplicate measurements.

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yielding a change in fluorescence intensity but no shift in λmax over a small distance range, typically approximately 5–10 Å (ref. 49).

To report on only the outward motion of TM6, we chose to label Leu266 mutated to cysteine instead of the native Cys265, because the former does not undergo inward-to-outward rotations after activation but points outward in both active and inactive structures (Supplementary Fig. 9a). On the basis of the inactive and active β 2AR crystal structures and dye simulations (Supplementary Fig. 9b), we speculated that a tryptophan residue introduced at the intracellu-lar end of TM5 (L230W) would quench the fluorescence of Atto655 in the inactive state, but the quenching would be considerably less in the active state, because the distance range and change between TM5 and TM6 that occurs after activation should be compatible with PET-induced quenching. By simulating the conformational ensembles of Atto655 bound to Cys266 in the inactive and active β 2AR structures, we found an average dye–Trp230 distance change of approximately 5 Å after receptor activation (Supplementary Fig. 9b), a result compatible with the reported quenching distance. We there-fore introduced mutations L230W and L266C into a minimal-cys-teine-background β 2AR33 and verified that the detergent-purified, Atto655-labeled β 2ARΔ 6 L230W L266C construct retained wild-type ligand binding properties (Supplementary Fig. 10).

When measuring the steady-state fluorescence emission of Atto655-labeled β 2ARΔ 6 L230W L266C, subsequently referred to as Atto-β 2AR, in the presence of saturating concentrations of epineph-rine, we observed an ∼ 30% increase in fluorescence intensity compared with that of ligand-free receptor, findings compatible with an out-ward motion of TM6 and an increased distance between Atto655 and the Trp230 quencher on TM5 (Fig. 6d and Supplementary Fig. 11a). Given the short distance over which PET quenching occurs, this observation probably reflects a TM6 displacement of ∼ 5 Å or more in ~30% of epinephrine-bound receptors. This result is consistent with the fraction of receptors in an active conformation observed in dou-ble electron–electron resonance spectroscopy studies31. Interestingly, we observed a much smaller difference (< 10%) between ligand-free and salmeterol-bound receptor (Fig. 6d and Supplementary Fig. 11a), thus suggesting that TM6 did not move sufficiently far from TM5 in salmeterol-bound β 2AR to decrease Atto655 quenching by Trp230. On the basis of the efficacy of salmeterol in G-protein activation (60% of isoproterenol; Supplementary Fig. 7c), we would expect a much larger change in Atto-β 2AR fluorescence in response to salme-terol if salmeterol and isoproterenol were to stabilize the same active state. The addition of Gs to salmeterol- and isoproterenol-bound Atto-β 2AR, as compared with ligand-free receptor, led to a 1.8- and 2.2-fold increase in fluorescence intensity, respectively (Fig. 6d, ‘+ Gs’ curves). This result is consistent with the G protein stabiliz-ing an active conformation in a larger fraction of receptor. Control measurements on a construct without the engineered L230W muta-tion showed little intensity change after addition of agonists alone or together with Gs (Supplementary Fig. 11b). In line with the differ-ence in TM6 outward motion between the Nb80- and Nb71-bound structures, the Atto response in the presence of Nb80 was much larger than that in the presence of Nb71, for both epinephrine and salmeterol (Supplementary Fig. 11c). This result was similar to our observations in the presence of Gs (Fig. 6b,d): when both ligand and Gs or nanobody were present, the TM6 receptor conformation was mainly stabilized by the intracellular binding protein and not the ligand. However, both of our fluorescence-based approaches in the absence of nanobody clearly suggested a difference in TM6 confor-mation between epinephrine- and salmeterol-bound receptors.

Discussionβ 2ARs expressed in airway smooth muscle and epithelial cells medi-ate bronchodilation and fluid clearance, respectively, and are thus well-established targets for the treatment of asthma and COPD50. The unusual pharmacological characteristics of salmeterol, including

the extremely high selectivity for β 2AR and the long duration of action, which can be attributed to its long aryloxyalkyl tail, make salmeterol among the most commonly prescribed LABAs for treat-ing asthma and COPD. Our structure reveals an additional site in the extracellular vestibule of β 2AR, the exosite, for the binding of the aryloxyalkyl tail, thus providing a structural basis for the prominent pharmacological action of salmeterol. It also resolves a long-stand-ing debate regarding the location of the exosite.

Our results also provide structural insights into the partial ago-nism and the biased signaling properties of salmeterol, which are attributed to its saligenin ethanolamine group bound in the ortho-steric binding pocket. Although the interactions with the receptor in the orthosteric binding pocket are very similar for the full agonist epinephrine and the partial agonist salmeterol, we observed subtle differences in hydrogen-bonding interactions. Most notable was the absence of interactions between salmeterol and Asn2936.55, as further supported by MD simulations. The meta-hydroxyl of epinephrine forms a hydrogen bond with Asn2936.55, which also forms a hydro-gen bond with Ser2045.43. Our mutagenesis studies suggest that this hydrogen-bond network may be important for stabilizing TM6 in a conformation necessary for efficient G-protein coupling as well as GRK phosphorylation and/or arrestin coupling. Therefore, the less extensive polar interactions between salmeterol and Asn2936.55 may contribute to the weaker efficacy of salmeterol in activating Gs and the near absence of β -arrestin recruitment, possibly because of inefficient coupling as a result of decreased GRK phosphorylation19.

The structure of salmeterol-bound β 2AR, compared with epineph-rine-bound β 2AR, revealed a smaller outward movement of TM6. Although this conformation implies a ‘partially active’ conformation of the receptor, it is probably imposed by the nanobody Nb71 rather than by the partial agonist salmeterol. Previous studies have suggested relatively weak allosteric coupling between the orthosteric site and the cytoplasmic site31. Thus, capturing the ligand-specific conforma-tion of β 2AR by protein crystallography is difficult. We used spectro-scopic approaches with two different reporter systems to interrogate the conformational changes of β 2AR associated with salmeterol, and showed that TM6 indeed did not achieve the same extent of outward motion as that achieved by the full agonist epinephrine. This find-ing correlates with previous single-molecule investigations of TM6 motion in the β 2AR33 and provides a structural basis for salmeterol’s weaker Gs efficacy and possibly ligand bias. Whether this mechanism applies to other GPCRs requires further investigation.

Online contentAny methods, additional references, Nature Research reporting summaries, source data, statements of data availability and asso-ciated accession codes are available at https://doi.org/10.1038/s41589-018-0145-x.

Received: 9 May 2018; Accepted: 6 September 2018; Published online: 16 October 2018

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AcknowledgementsThis work was supported by National Institutes of Health grant R01NS028471 (B.K.K.), Canadian Institute for Health Research foundation grant FDN-148431 (M.B.), an American Heart Association Postdoctoral fellowship (17POST33410958; M.M.) and Predoctoral Fellowship (13PRE17110027; J.P.M.), a studentship from the FRQ-S (L.-P.P.), the NIH Pharmacological Sciences Training Program (T32GM007767; J.P.M.) and the National Institutes of Health MIRA 1R35GM128641-01 (C.Z.). B.K.K. is supported by the Chan Zuckerberg Biohub. M.B. is supported as a Canada Research Chair in Signal Transduction and Molecular Pharmacology. The authors thank J. Gullingsrud for assistance with MD software.

Author contributionsC.Z. and Y.Z. expressed and purified the receptor and nanobody for crystallography studies, collected X-ray diffraction data and solved the crystal structure. M.M. developed the Atto655 reporter system; purified and labeled receptors used in fluorescence studies; collected spectroscopic data; and performed radioactive ligand binding assays. L.-P.P. generated mutants of N293 and S204. E.v.d.W. and L.-P.P. performed Gs-activation and β -arrestin2-recruitment BRET assays under supervision from M.B. J.P.M. performed Octet RED experiments under supervision from R.K.S. J.P.G.L.M.R. performed modeling and sampling of the Atto655 dye on the receptor structures. T.J.M. and R.O.D. performed and analyzed MD-simulation studies. R.O.D. and D.E.S. oversaw MD simulations and analysis. E.P. generated the nanobody library and performed the initial selections. J.S. supervised nanobody production. W.I.W. supervised and assisted with the structure refinement. M.M., C.Z. and B.K.K. interpreted data, made figures and wrote the manuscript. B.K.K. provided overall project supervision.

Competing interestsThe BRET-based biosensors used in the present study are licensed to Domain Therapeutics but are freely available from M.B. for noncommercial academic use. M.B. is the chair of the Scientific Advisory Board of Domain Therapeutics. B.K.K. is a cofounder of and consultant for ConfometRx.

Additional informationSupplementary information is available for this paper at https://doi.org/10.1038/s41589-018-0145-x.

Reprints and permissions information is available at www.nature.com/reprints.

Correspondence and requests for materials should be addressed to C.Z. or B.K.K.

Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

© The Author(s), under exclusive licence to Springer Nature America, Inc. 2018

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MethodsReceptor constructs. All constructs had an N-terminal FLAG tag. The T4L-β 2AR-Δ ICL3 construct consisted of an N-terminal T4 lysozyme fusion to β 2AR(29–365)-Δ ICL3, i.e., Δ (235–263) with mutations M96T, M98T and N187E. The β 2AR-PN1 construct consisted of the β 2(1–24)-TEV-β 2(25–365)-3C-β 2(366–413) construct, with TEV and 3C indicating tobacco etch virus (TEV) protease and 3C protease cut sites, respectively. Additionally, mutations M96T, M98T, C378A, N187E and C406A were introduced, as previously described46. The β 2ARΔ 6 L230W L266C construct consisted of a full-length wild-type receptor with a minimal cysteine background33 (C77V, C265A, C327S, C341L, C378A and C406A) as well as mutations L230W and L266C and a C-terminal hexahistidine tag.

Expression, purification and labeling of β2AR constructs. Receptor constructs were expressed in Sf9 insect-cell cultures infected with recombinant baculovirus (BestBac, Expression Systems) and solubilized in n-dodecyl-β -d-maltoside (DDM) according to methods described previously46. The solubilized receptor was purified through M1 FLAG chromatography followed by alprenolol–Sepharose chromatography to remove nonfunctional receptor46. A second M1 FLAG chromatography step was performed such that the receptor-bound alprenolol could be removed for ligand-free protein or exchanged for salmeterol.

For the T4L-β 2AR-Δ ICL3, after the protein was eluted from the M1 resin, the FLAG epitope tag of T4L-β 2AR-Δ -ICL3 was removed by treatment with TEV protease (Invitrogen) for 3 h at room temperature or overnight at 4 °C. When necessary, a 50-kDa MWCO Vivaspin concentrator (GE Healthcare) was used to concentrate the receptor. The β 2AR constructs used for fluorescence studies were concentrated and flash-frozen in the presence of 20% glycerol at a final concentration of 200 μ M. Aliquots were then stored at –80 °C until use.

Full-length PN1-β 2AR was labeled with monobromobimane as previously described51. Briefly, FLAG pure receptor (∼ 2 µ M) was incubated overnight on ice with 100 μ M TCEP and 20 μ M monobromobimane (Thermo Fisher Scientific). Subsequently, 5 mM cysteine was added to quench the labeling reaction, and alprenolol–Sepharose chromatography was performed as described above. A similar procedure was followed for labeling β 2ARΔ 6 L230W L266C with Atto655-iodoacetamide (Atto-TEC).

G-protein expression and purification. Wild-type Gs heterotrimer was expressed and purified as previously described33. After purification, the protein was dialyzed into 20 mM HEPES, pH 7.5, 100 mM NaCl, 0.02% DDM, 100 μ M TCEP and 20 μ M GDP, concentrated, and flash-frozen in the presence of 20% glycerol at a final concentration of 200 μ M. Aliquots were then stored at –80 °C until use.

Preparation of Nb71 and Nb80. The recombinant Nb71 was generated in the same way as Nb80 (ref. 25). These two nanobodies were screened from an identical library of single-chain nanobody clones after immunization of a llama with purified agonist-bound β 2AR reconstituted at high density into phospholipid vesicles. Nb71 was expressed in Escherichia coli and purified by nickel-affinity chromatography in the same manner as Nb80 (ref. 25). The protein was then further purified by cation exchange with a Mono-S column (GE Healthcare); the protein was loaded in 20 mM NaCl and 20 mM MES, pH 6.0, and eluted with a linear gradient from 50 to 500 mM NaCl. To minimize severe precipitation of Nb71 over time, the purified protein was stored at a concentration below 5 mg/mL in 20 mM HEPES and 1 M NaCl.

Crystallization. Salmeterol-bound receptor (~40 mg/mL) was incubated with a 5.5-fold molar excess of Nb71 (~50 mg/mL) for 1 h on ice. Size-exclusion chromatography was then performed to remove free Nb71. The purified complex was concentrated to ~60 mg/mL for crystallization with the lipid cubic phase method as previously described36. The protein complex was reconstituted in mono-olein containing 10% cholesterol at a 1:1.5 protein/lipid ratio (wt/wt). Drops of reconstituted protein–lipid mixture (30 nL) were deposited in each well of a 96-well glass sandwich plate (Molecular Dimensions). The drop was then overlaid with 650 nL of precipitant, and the wells were sealed with a glass coverslip. Diffraction-quality crystals were grown at 20 °C in 31–34% PEG 400, 100 mM HEPES, pH 7.5, and 1% 1,2,3-heptanetriol after 3 d of incubation at 20 °C.

Data collection, structure determination and analysis. Crystals were harvested and frozen in liquid nitrogen directly without use of additional cryoprotectant. Diffraction data from 24 different crystals were measured with the GM/CA-CAT microfocus beam at 23-ID-D (Advanced Photon Source, Argonne National Labs). The data were processed with HKL2000, and the structure was solved by molecular replacement with Phaser. Further model rebuilding was performed with Coot, and the structure was refined with Phenix. The validation of the final structural model was performed with MolProbity. The overall MolProbity score was 1.35. In Ramachandran analysis, 96.8% of atoms were in favored regions, and 3.2% of atoms were in allowed regions. Data processing and refinement statistics are shown in Supplementary Table 2.

The structure of the inactive β 2AR (PDB 2RH1); β 2AR in complex with Nb80 (PDB 3P0G); β 2AR in complex with Gs (PDB 3SN6); and chain A of sabultamol bound to β 1AR (PDB 2Y04) were used for structural alignments in PyMOL, on the

basis of Cα only. The secondary structure of β 2AR ICL2 was assigned with PyMOL and DSSP.

Reconstitution of β2AR in HDL particles for Octet RED measurements. The β 2AR was reconstituted in high-density lipoprotein (HDL) particles through established methods46. The scaffold protein ApoA-I used for reconstitution was purified as previously described46. Purified ApoA-I was biotinylated for 30 min at room temperature with NHS–PEG4–biotin (Thermo) at a 1:1 molar ratio in a buffer composed of 20 mM HEPES, pH 8.0, 100 mM NaCl, 1 mM EDTA and 5 mM sodium cholate. After the labeling reaction, unreacted biotin was quenched by the addition of Tris-HCl, pH 8.0, to a final concentration of 20 mM. Biotinylated protein was separated from free biotin by size-exclusion chromatography on a Superdex 75 HR 10/30 column. FLAG-tagged β 2AR was incorporated into HDL particles with biotinylated ApoA-I, and receptor-containing HDL particles were isolated through M1 anti-FLAG immunoaffinity chromatography.

Nanobody binding to β 2AR in biotinylated HDL particles was monitored with an Octet RED biolayer interferometry system (Pall FortéBio) with streptavidin-coated biosensors (Pall FortéBio). Sensors were hydrated for at least 10 min at room temperature in assay buffer (20 mM Tris-HCl, pH 7.4, 136 mM NaCl, 2.7 mM KCl, 1 mM EDTA, 0.02% (wt/vol) ascorbic acid and 0.05% (wt/vol) BSA), then incubated with biotinylated β 2AR–HDL (~100 nM) for 10 min at room temperature before the sensors were loaded onto the Octet RED instrument. All steps on the Octet RED instrument were performed at 25 °C with the assay plate shaking at 1,000 r.p.m. After an initial baseline reading, sensors were dipped into wells containing assay buffer with a saturating concentration of agonist (100 µ M epinephrine, 0.1 µ M BI-167107 or 1 µ M salmeterol) and incubated for 20 min to equilibrate β 2AR with the agonist. The sensors were transferred to wells containing assay buffer plus agonist and nanobody (1, 3.2, 10, 32, 100 or 320 nM) for 5 min to monitor nanobody association. Nanobody dissociation was then monitored by transferring the sensors to wells containing assay buffer plus agonist for 30 min. Nonspecific nanobody binding at each concentration was measured in a parallel experiment in which sensors were loaded with empty HDL. Buffer-only controls were also included in each experiment to correct for baseline drift. Data were first analyzed in Octet Data Analysis 7.0 software (Pall FortéBio) to remove baseline and nonspecific binding, and the processed data were exported to Prism 6 (GraphPad) for curve fitting. All association and dissociation curves were fit with single-phase exponential association or decay.

BRET measurements for Gs activation, β-arrestin recruitment and β2AR conformational changes in live cells. HEK293T cells used for the BRET assays were grown in Dulbecco’s modified Eagle’s medium supplemented with 10% newborn calf serum, at 37 °C with 5% CO2. Cells were detached with trypsin–EDTA and transfected with 2.5 μ g of total DNA per 106 cells, with linear polyethyleneimine (Polysciences) as a transfecting agent, at a ratio of 3:1 polyethyleneimine/DNA. Gs activation was evaluated with Gs-117-RLucII/Gβ 1/Gγ 1-GFP10 (ref. 42), and β -arrestin recruitment was evaluated with CAAX-rGFP/β -arr2-RLucII43 sensors, in the presence of wild-type 3HA-β 2AR or mutant receptors. The conformational changes were detected with the NY-β 2AR sensor44. Directly after transfection, cells were plated in white 96-well plates (Greiner) at a density of 50,000 cells/well and incubated for 48 h. The plates were then washed with PBS, and assay buffer (Hank’s balanced salt solution) was added. Cells were stimulated with the ligands during 5 or 15 min for evaluation of Gs activation or β -arrestin recruitment and conformational changes, respectively. Coelenterazine 400a (2.5 μ M) was added 5 min before the reads. BRET was monitored with a TriStar LB942 microplate reader (Berthold) equipped with a 410/80-nm donor filter and a 515/40-nm acceptor filter (for Gs activation and β -arrestin recruitment) or a 485/20-nm donor filter and a 530/25-nm acceptor filter (for receptor conformational changes). The BRET ratio was calculated by dividing the acceptor emission by the donor emission. The data were analyzed in Prism (GraphPad) by using ‘dose-response- stimulation log(agonist) vs normalized response- variable slope’ with the constraint of sharing the Hill slope across all datasets.

Molecular dynamics simulations. We simulated the β 2AR bound to the partial agonist salmeterol as well as the full agonist epinephrine. We initiated these simulations from crystal structures of the receptor bound to each of these ligands. Each of the crystal structures that we used includes a nanobody that binds to the intracellular side of the receptor and stabilizes an active or intermediate receptor state: Nb71 for salmeterol and Nb6B9 for epinephrine29.

We performed three simulations of β 2AR–salmeterol–Nb71 and three simulations of β 2AR–epinephrine–Nb6B9. Each of the crystallized constructs was a β 2AR-T4L fusion protein, with T4L replacing the receptor’s N terminus. T4L was omitted from all simulations, whereas all other resolved residues were included. Most of ICL3 was absent in all simulations, because it was deleted from the crystallized construct in β 2AR–salmeterol–Nb71 and β 2AR–epinephrine–Nb6B9. Because the simulations were performed before the published coordinates had been finalized, they were initiated by using coordinates that differed very slightly from the published ones (r.m.s. deviation below 0.3 Å, computed over all resolved receptor Cα atoms); these differences were much smaller than the typical motions of the atoms in simulation. In all simulations, a palmitoyl group that was not

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resolved but was presumed to be present in the crystallized constructs was added to Cys341 with Maestro (Schrödinger).

The β 2AR was embedded in a hydrated lipid bilayer in all simulations; all atoms (including those in lipids and water) were represented explicitly. Hydrogen atoms were added to the crystal structures with Maestro, as described previously, and receptor chain termini were capped with neutral groups (acetyl and methylamide). Titratable residues other than Glu1223.41 were left in their dominant protonation states at pH 7.0. Glu1223.41, which faces the lipid bilayer, was neutral (protonated) in all simulations.

The prepared protein structures were inserted into an equilibrated bilayer solvated with 0.15 M NaCl, through a previously described protocol52. The bilayer consisted of palmitoyl-oleoyl-phosphatidylcholine, with 24% palmitoyl-oleoyl-phosphatidylserine in the inner-membrane leaflet. The simulated systems initially measured approximately 71 × 71 × 115 Å3 and contained approximately 140 lipid molecules, 12,000 water molecules, 32 chloride ions and 40 sodium ions.

We also performed three simulations of β 2AR–salmeterol with the nanobodies removed (but otherwise starting from the same structures as above). The simulation setup was as described above, except that the removal of the nanobody allowed for the simulated volume to be somewhat smaller. These simulations initially measured approximately 69 × 69 × 85 Å3 and contained approximately 120 lipid molecules, 7,200 water molecules, 19 chloride ions and 30 sodium ions.

We used the CHARMM-h force field for proteins. We used the CHARMM36 lipid force field along with standard CHARMM salt-ion parameters and the CHARMM TIP3P model for water. The parameters for palmitoyl-cysteine were as described previously27,52, and the parameters for epinephrine were obtained by adaptation of previously published parameters for isoproterenol53. The parameters for salmeterol were obtained from the ParamChem server. Full parameter sets are available upon request.

Each simulation consisted of a 50-ns equilibration run followed by a longer production run. Systems were equilibrated in the NPT ensemble (310 K, 1 bar, Martyna–Tuckerman–Klein–Nosé–Hoover chain-coupling scheme using a multigrator, with initial velocities sampled from the Boltzmann distribution and with 5 kcal mol−1 Å−2 harmonic position restraints applied to the protein and ligand atoms, which were tapered off linearly over 50 ns. Production simulations used the same integrator, pressure and temperature, and were initiated from the final snapshot of the corresponding equilibration simulation. All simulations were performed on an Anton 1 computer54.

All bond lengths to hydrogen atoms were constrained with M-SHAKE. An r-RESPA integrator was used with a time step of 2.5 fs, and long-range electrostatics were computed every 7.5 fs. Long-range electrostatics were computed in reciprocal space with the u-series method55.

Simulation snapshots were saved every 180 ps. For the purposes of evaluating the fraction of time, a hydrogen bond was formed in simulation; such a bond was considered to be formed in snapshots in which the relevant nonhydrogen atoms were within 3.0 Å of one another. Because the bound ligands shifted in pose during the later parts of certain simulations (possibly as a result of motion of the nanobodies, which are not held in place by crystallographic contacts), we used the first microsecond of each simulation for these analyses. Each error bar was calculated as s.e.m. across three simulations.

Dye modeling. The structure of the iodoacetamide derivative of the Atto655 dye (Atto-TEC) was obtained from PubChem (CID 16218785) and optimized with Avogadro and the GAFF force field. The structures of β 2AR in the active (PDB 3SN6) and inactive conformations (PDB 2RH1) were retrieved from the RCSB Protein Data Bank and mutated (L230W and L266C) with PyMOL. The parameters for the dye molecule attached to a cysteine residue were obtained with PRODRG and manually edited to enforce the planarity of the ring system of the dye. Crystallography and NMR System (CNS) software, version 1.3 (ref. 56), was used to attach the dye to L266C and simulate its positions through rigid-body docking and multitrial simulated annealing, respectively, as previously performed by Brunger and co-workers57. The entire receptor structure was kept rigid except for the side-chain atoms of L230W and L266C. Distances between the centers of mass of the tryptophan side-chain atoms and the center of mass of the ring system of the dye were calculated for each generated model (300 for each conformation of β 2AR).

Fluorescence measurements on purified receptor in detergent solution. Bimane-labeled and Atto-labeled receptors were used at respective concentrations of 0.2 µ M

and 0.1 µ M in buffer containing 20 mM HEPES, pH 7.4, 100 mM NaCl, 0.02% (wt/vol) DDM and 0.002% (wt/vol) cholesterol hemisuccinate. Salmeterol and epinephrine stocks were prepared as 50 mM and 100 mM solutions in DMSO and added at the indicated final concentrations. Ligand concentrations were chosen to achieve saturation of detergent-solubilized receptor. To avoid any nonspecific vehicle effects, care was taken to obtain the same final concentration of DMSO in all samples. Where indicated, Gs was added to a final concentration of 2 µ M. To allow for efficient ligand binding and full equilibration, samples were incubated at the final concentrations used for 1 h in the dark before measurements. Fluorescence data were collected in a quartz cuvette with 500 µ L of sample in FluorEssence v3.8 software on a Fluorolog instrument (Horiba) in photon-counting mode. Bimane fluorescence was measured by excitation at 370 nm with excitation and emission bandwidth passes of 4 nm, and emission spectra were recorded from 410 to 510 nm in 1-nm increments and 1-s integration time. Atto fluorescence was measured by excitation at 650 nm with excitation and emission bandwidth passes of 5 nm, and emission spectra were recorded from 660 to 730 nm in 1-nm increments and 1-s integration time. Measurements were performed in triplicate.

Radioligand binding assays. Binding curves were obtained by incubation of DDM-purified wild-type and bimane- and Atto655-labeled receptors in the presence of M1 FLAG–Sepharose and 2 mM Ca2+, under shaking at room temperature for 1.5 h. The antagonist [3H]dihydroalprenolol (PerkinElmer) was used to obtain saturation binding curves. Competition binding measurements for salmeterol and epinephrine were performed in a similar way, with [3H]dihydroalprenolol at a concentration slightly above the Kd, as determined by saturation binding. Nonspecific binding was accounted for by measurements in the presence of 10 μ M cold alprenolol. Beads were harvested with a 48-well Brandell harvester and counted in a LS6000TA scintillator (Beckman) with Cytoscint (MP Biomedical).

Statistical analysis. The statistical significance of hydrogen-bond duration from MD trajectories (Fig. 5a) was assessed with one-sided Welch’s unequal variance t tests. The statistical significance of the binding affinities (Supplementary Table 1) was assessed with ordinary one-way ANOVA (Tukey) test at a multiplicity-adjusted P-value cutoff of 0.05. The statistical significance of the fitted mean log(Ki) values (Supplementary Fig. 1) was assessed with a one-way ANOVA Tukey test at a P-value cutoff of 0.05 (adjusted for multiple comparisons).

Reporting Summary. Further information on research design is available in the Nature Research Reporting Summary linked to this article.

Data availabilityAtomic coordinates and structure factors for the crystal structure have been deposited in the Protein Data Bank under accession code PDB 6CSY. Other data and results are available upon request.

References 51. Rosenbaum, D. M. et al. GPCR engineering yields high-resolution

structural insights into β 2-adrenergic receptor function. Science 318, 1266–1273 (2007).

52. Dror, R. O. et al. Identification of two distinct inactive conformations of the β 2-adrenergic receptor reconciles structural and biochemical observations. Proc. Natl. Acad. Sci. USA 106, 4689–4694 (2009).

53. Dror, R. O. et al. Pathway and mechanism of drug binding to G-protein-coupled receptors. Proc. Natl. Acad. Sci. USA 108, 13118–13123 (2011).

54. Shaw, D. E. et al. Millisecond-scale molecular dynamics simulations on Anton. Proc. Conf. High Perform. Comput. Netw. Storage Anal. (pp. 1–11. ACM, Portland, Oregon, USA, 2009).

55. Shaw, D. E. et al. Anton 2: raising the bar for performance and programmability in a special-purpose molecular dynamics supercomputer. Sc14: Int. Conf. High Perform. Comput., Netw. Storage Anal. (pp. 41–53. IEEE, Hoboken, New Jersey, USA, 2014).

56. Brunger, A. T. Version 1.2 of the Crystallography and NMR system. Nat. Protoc. 2, 2728–2733 (2007).

57. Choi, U. B. et al. Single-molecule FRET-derived model of the synaptotagmin 1-SNARE fusion complex. Nat. Struct. Mol. Biol. 17, 318–324 (2010).

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Corresponding author(s): Cheng Zhang, Brian Kobilka

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Data collection Octet Red data were collected using OctetRED biolayer interferometry system (Pall FortéBio). Fluorescence data were collected using FluorEssence v3.8 (Horiba). Molecular dynamics simulations were performed on Anton (Assoc Computing Machinery, New York).

Data analysis Octet data were processed using Octet Data Analysis 7.0 software (Pall FortéBio) to remove baseline and nonspecific binding and further analyzed in Prism 6 (GraphPad) for curve fitting. Crystallography data were processed with HKL2000 and the structure solved by molecular replacement using Phaser. Further model rebuilding was performed by using Coot and the structure was refined with Phenix. The validation of the final structural model was performed using Molprobity. BRET data were analyzed in Prism 7 (GraphPad). For simulations, a palmitoyl group not resolved but presumed to be present in the crystallized constructs was added to Cys341 using Maestro (Schrödinger LLC, New York, NY). The structure of the iodoacetamide derivative of the Atto655 dye was optimized using Avogadro and the GAFF force field. Parameters for the dye molecule attached to a cysteine residue were obtained using PRODRG. The Crystallography and NMR System (CNS) software, version 1.3, was used to attach the dye to L266C and simulate its positions. Radioligand binding data were analyzed in Prism 7 (GraphPad) using "One site -- Total and nonspecific binding" for saturation binding and "Nonlinear Regression - One site - Fit Ki" fitting for competition binding. Figures for structures were made in PyMOL v1.8.4.1 (Schrödinger). The 2-D representation ligand plot figure was generated using LigPlot+. Fluorescence data were analyzed in Prism 7 (GraphPad).

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Sample size No animal studies were performed in this study. No statistical method was used to predetermine sample size. For all measurements, the experimental methods are conventional and widely used, and detailed and cited in the methods section. According to general convention for these methods, 3 to 5 replicate measurements are sufficient to obtain statistical significance (calculation of mean and standard deviation or standard error of the mean) to allow to draw scientific conclusions.

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Obtaining unique materials Nanobody 71, a camelid antibody can be obtained from Jan Steyaert, Structural Biology Brussels, Vrije Universiteit Brussel, Brussels, Belgium

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AntibodiesAntibodies used Nanobody 71 is a camelid antibody generated by the laboratory of Jan Steyaert with protein produced in the laboratory of Brian

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Cell line source(s) The Sf9 insect cell line used was obtained from Expression Systems and is a clonal isolate derived from the parental Spodoptera frugiperda cell line IPLB-Sf-21-AE. The HEK293 cell line used in this study is the one in which BRET-based biosensors have been developed in Dr Michel Bouvier’s laboratory.

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